Method of treating or retarding the development of blindness

ABSTRACT

A method for treating an ocular disorder characterized by the defect or absence of a normal gene in the ocular cells of a human or animal subject involves administering to the subject by subretinal injection an effective amount of a recombinant adeno-associated virus carrying a nucleic acid sequence encoding the normal gene under the control of a promoter sequence which expresses the product of the gene in the ocular cells. The ocular cells are preferably retinal pigment epithelial (RPE) cells, and the gene is preferably an RPE-specific gene, e.g., RPE65. The promoter is one that can express the gene product in the RPE cells. Compositions for subretinal administration are useful in this method.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of International PatentApplication No. PCT/US02/11314, filed Apr. 11, 2002, which claims thebenefit of the priority of U.S. Provisional Patent Application No.60/283,766, filed Apr. 13, 2001, now abandoned.

BACKGROUND OF THE INVENTION

[0002] The invention relates generally to the use of recombinant virusesto deliver a desired transgene to retinal pigment epithelial cells ofpatients suffering from retinal degenerative diseases.

[0003] The relationship between the neurosensory photoreceptors and theadjacent retinal pigment epithelium (RPE) controls not only normalretinal function, but also the pathogenesis of hereditary retinaldegenerations. Recent progress has identified the molecular bases forprimary photoreceptor diseases, such as retinitis pigmentosa (Dryja, T.P., et al. 1990 Nature 343, 364-366; Farrar, G. J., et al. 1991 Nature354, 478-480; and McLaughlin, M. E. et al, 1993 Nature Genetics 4,130-134). Similarly the molecular bases for RPE diseases that causephotoreceptor blindness, such as child-onset severe retinal dystrophy,Leber's congenital amaurosis, and Best macular dystrophy, have beenidentified (Gu, S. -M., et al. 1997 Nature Genetics 17, 194-197;Marlhens, F., et al. 1997 Nature Genetics 17, 139-141; Petrukin, K., etal. 1998 Nature Genet 19, 241-247; and D'Cruz, P., et al.2000 Hum. Mol.Genet. 9, 645-651). Despite these reported scientific advances,effective therapy for human retinal degenerations is still lacking.

[0004] Retinal gene therapy has been considered a possible therapeuticoption for man. For example, U.S. Pat. No. 5,827,702 refers to methodsfor generating a genetically engineered ocular cell by contacting thecell with an exogenous nucleic acid under conditions in which theexogenous nucleic acid is taken up by the cell for expression. Theexogenous nucleic acid is described as a retrovirus, an adenovirus, anadeno-associated virus or a plasmid. See, also, International PatentPublications WO 00/15822, published Mar. 23, 2000 and WO 98/48097,published Oct. 29, 1998.

[0005] A review of gene therapy efforts to date indicates that suchefforts have focused mainly on slowing down retinal degeneration inrodent models of primary photoreceptor diseases. Normal genes andmutation-specific ribozymes delivered to photoreceptors have prolongedthe lifetime of these cells otherwise doomed for apoptotic cell death(Bennett, J., et al. 1996 Nat. Med. 2, 649-654; Bennett, J., et al. 1998Gene Therapy 5, 1156-1164; Kumar-Singh, R. & Farber, D., 1998 Hum. Mol.Genet. 7, 1893-900; Lewin, A. S., et al. 1998 Nat. Med. 4, 967-971; Ali,R., et al. 2000 Nat. Genet. 25, 306-310; Takahashi, M. et al, 1999 JVirol. 73, 7812-6; Lau, D., et al.2000 Invest. Ophthalmol. Vis. Sci. 41,3622-3633; and LaVail, M. M., et al. 2000 Proc Natl Acad Sci USA 97,11488-11493).

[0006] Retinal gene transfer of a reporter gene, green fluorescentprotein, using a recombinant adeno-associated virus was demonstrated innormal primates (Bennett, J., et al. 1999 Proc. Natl. Acad. Sci. USA 96,9920-9925). However, an as-yet unmet goal of research is the restorationof vision in a blinding disease of animals, particularly humans andother mammals, caused by genetic defects in RPE and/or photoreceptorcells.

[0007] There remains a need in the art for methods for effectivelytreating humans and other mammals or other animals suffering fromblindness due to genetic defects or deficiencies, so as to restoresufficient vision to enable the subject to function in response tovisual cues.

BRIEF SUMMARY OF THE INVENTION

[0008] In one aspect, the invention provides a method for treating anocular disorder in a human or animal subject characterized by the defector absence of a normal gene in the ocular cells. The method includesadministering to the subject by subretinal injection an effective amountof a recombinant adeno-associated virus carrying a nucleic acid sequenceencoding the normal gene under the control of a promoter sequence whichexpresses the product of the gene in the ocular cells.

[0009] In another aspect, the invention provides a method for treatingan ocular disorder in a human or animal subject characterized by thedefect or absence of a normal gene in the retinal pigment epithelial(RPE) cells of the subject. The method involves administering to thesubject by subretinal injection an effective amount of a recombinantvirus carrying a nucleic acid sequence encoding a normal retinal pigmentepithelial (RPE) cell-specific gene under the control of a promotersequence which expresses the product of the gene in RPE cells. In oneembodiment, the gene is the RPE65 gene.

[0010] In another aspect, the invention provides a method for treatingLeber congenital amaurosis in a subject by administering to the subjectby subretinal injection an effective amount of a recombinant viruscarrying a nucleic acid sequence encoding a normal gene under thecontrol of a promoter sequence which expresses the product of the genein ocular cells, wherein the cells contain a mutated version of thegene. Expression of the normal gene provides to the cells the productnecessary to restore or maintain vision in the subject. In oneembodiment, the cells are RPE or photoreceptor cells, and the promotersare cell-specific promoters.

[0011] In still another embodiment, the invention provides a compositionfor treatment of an ocular disorder characterized by the defect orabsence of a normal gene in the ocular cells of the subject. Suchcompositions comprise effective amounts of a recombinantadeno-associated virus carrying a nucleic acid sequence encoding thenormal gene under the control of a promoter sequence which expresses theproduct of the gene in the ocular cells, formulated with a carrier andadditional components suitable for subretinal injection. In oneembodiment, the normal gene is RPE65.

[0012] Other aspects and advantages of the present invention aredescribed further in the following detailed description of the preferredembodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The invention provides a method for treating an ocular disorderin a human, other mammalian or other animal subject. In particular, theocular disorder is one which involves a mutated or absent gene in aretinal pigment epithelial cell or a photoreceptor cell. The method ofthis invention comprises the step of administering to the subject bysubretinal injection an effective amount of a recombinant virus carryinga nucleic acid sequence encoding an ocular cell-specific normal geneoperably linked to, or under the control of, a promoter sequence whichdirects the expression of the product of the gene in the ocular cellsand replaces the lack of expression or incorrect expression of themutated or absent gene.

[0014] A. The Ocular Disorder

[0015] In particular, this method is useful for the treatment and/orrestoration of at least partial vision to subjects that have lost visiondue to ocular disorders, such as RPE-associated retinopathies, which arecharacterized by a long-term preservation of ocular tissue structuredespite loss of function and by the association between function lossand the defect or absence of a normal gene in the ocular cells of thesubject. A variety of such ocular disorders are known, such as childhoodonset blinding diseases, retinitis pigmentosa, macular degeneration, anddiabetic retinopathy, as well as ocular blinding diseases known in theart. It is anticipated that these other disorders, as well as blindingdisorders of presently unknown causation which later are characterizedby the same description as above, may also be successfully treated bythis method. Thus, the particular ocular disorder treated by this methodmay include the above-mentioned disorders and a number of diseases whichhave yet to be so characterized. For purposes of illustration of thisinvention, the particular ocular disorder being treated in the examplesis Leber congenital amaurosis, which affects humans. However, thisinvention is not limited to the treatment of that disorder alone.

[0016] Leber congenital amaurosis (LCA) is a severe childhood-onsetblinding disease which can be caused by mutations in the retinal pigmentepithelium (RPE)-specific gene, RPE65. A naturally-occurring largeanimal model of an analogous severe disease of retinal degenerations isthe RPE65 mutant dog. LCA causes near total blindness from early inlife. Among the molecular causes of LCA are mutations in the geneencoding an RPE protein, RPE65. RPE65 is an evolutionarily-conserved 65kDa membrane-associated protein (Redmond, T. & Hamel, C. 2000 Meth.Enzymol. 317, 705-724 and Bavik, C. et al, 1992 J. Biol. Chem. 267,23035-23042), which is important in retinoid metabolism (Saari, J. 2000Invest Ophthalmol Vis Sci 41, 337-348; Ma, J. -X. et al, 1998 J BiolChem 1443, 255-261; and Simon, A. et al, 1995 J Biol Chem 270,1107-1112). Currently there is no treatment for LCA and related earlyonset retinal degenerative diseases.

[0017] RPE65 deficiency in mice results in accumulation ofall-trans-retinyl esters, undetectable levels of rhodopsin, rodphotoreceptor dysfunction, inclusions in the RPE, and slow retinaldegeneration. The compound 9-cis-retinal can restore visual pigment andfunction in RPE65-deficient mice (Redmond, T., et al. 1998 Nat. Genet20, 344-351 and Van Hooser, J. P., et al. 2000 Proc. Natl Acad Sci USA97, 8623-8628).

[0018] The RPE65 mutant dog shows early and severe visual impairmentcaused by a homozygous 4 bp-deletion in the RPE65 gene. The deletionresults in a frame shift leading to a premature stop codon, eliminatingmore than two-thirds of the wildtype polypeptide. Histopathology inhomozygotes shows prominent RPE inclusions and slightly abnormal rodphotoreceptor morphology present within the first year of life, andslowly progressive photoreceptor degeneration in older dogs. See, e.g.,Wrigstad, A. Hereditary Dystrophy of the Retina and the Retinal PigmentEpithelium in a Strain of Briard Dogs: A Clinical, Morphological andElectrophysiological Study. Linkoping University Medical Dissertations(1994); Narfstrom, K. et al, 1989 Brit J Ophthalmol. 73, 750-756; andAguirre, G., et al. 1998 Mol. Vis. 4, 23.

[0019] B. Vectors for Use in the Method According to the variousembodiments of the present invention, a variety of known nucleic acidvectors may be used in these methods, e.g., recombinant viruses, such asrecombinant adeno-associated virus (AAV), recombinant adenoviruses,recombinant retroviruses, recombinant poxviruses, and other knownviruses in the art, as well as plasmids, cosmids and phages, etc. Awealth of publications known to those of skill in the art discusses theuse of a vaiety of such vectors for delivery of genes (see, e.g.,Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1989; Kay, M. A. et al, 2001 Nat. Medic., 7(1):33-40;and Walther W. and Stein U., 2000 Drugs, 60(2):249-71). In oneembodiment of this invention the vector is a recombinant AAV carrying awildtype (i.e., normal) version of a selected transgene-encoding cDNAdriven by a promoter that expresses the product of the wildtype cDNA inselected ocular cells of the affected subject. Methods for assembly ofthe recombinant vectors are well-known (see, e.g., International PatentPublication WO 00/15822, published Mar. 23, 2000 and other referencescited herein). To exemplify the methods and compositions of thisinvention, the presently preferred vector, a recombinant AAV isdescribed in detail.

[0020] 1. AAV Vectors

[0021] Adeno-associated viruses are small, single-stranded DNA viruseswhich require helper virus to facilitate efficient replication (K. I.Berns, Parvoviridae: the viruses and their replication, p. 1007-1041, inF. N. Fields et al., Fundamental Virology, 3rd ed., vol. 2,(Lippencott-Raven Publishers, Philadelphia, Pa.) (1995)). The 4.7 kbgenome of AAV is characterized by two inverted terminal repeats (ITR)and two open reading frames which encode the Rep proteins and Capproteins, respectively. The Rep reading frame encodes four proteins ofmolecular weight 78 kD, 68 kD, 52 kD and 40 kD. These proteins functionmainly in regulating AAV replication and rescue and integration of theAAV into a host cell's chromosomes. The Cap reading frame encodes threestructural proteins of molecular weight 85 kD (VP 1), 72 kD (VP2) and 61kD (VP3) (Berns, cited above) which form the virion capsid. More than80% of total proteins in AAV virion comprise VP3.

[0022] Flanking the rep and cap open reading frames at the 5′ and 3′ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp ofwhich are capable of forming Y- or T-shaped duplex structures. The twoITRs are the only cis elements essential for AAV replication, rescue,packaging and integration of the AAV genome. There are two conformationsof AAV ITRs called “flip” and “flop”. These differences in conformationoriginated from the replication model of adeno-associated virus whichuses the ITR to initiate and reinitiate the replication (R. O. Snyder etal, 1993, J Virol., 67:6096-6104 (1993); K. I. Berns, 1990Microbiological Reviews, 54:316-329). The entire rep and cap domains canbe excised and replaced with a therapeutic or reporter transgene (B. J.Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp.155-168 (1990)).

[0023] AAVs have been found in many animal species, including primates,canine, fowl and human (F. A. Murphy et al., “The Classification andNomenclature of Viruses: Sixth Report of the International Committee onTaxonomy of Viruses”, Archives of Virology, (Springer-Verlag, Vienna)(1995)). Six primate serotypes have been reported (AAV1, AAV2, AAV3,AAV4, AAV5 and AAV6). The AAV ITR sequences and other AAV sequencesemployed in generating the minigenes, vectors, and capsids, and otherconstructs used in the present invention may be obtained from a varietyof sources. For example, the sequences may be provided by AAV type 5,AAV type 2, AAV type 1, AAV type 3, AAV type 4, AAV type 6, or other AAVserotypes or other densoviruses, including presently identified humanAAV types and AAV serotypes yet to be identified. Similarly, AAVs knownto infect other animals may also provide these ITRs employed in themolecules or constructs of this invention. Similarly, the capsids from avariety of serotypes of AAV may be “mixed and matched” with the othervector components. See, e.g., International Patent Publication No.WO01/83692, published Nov. 8, 2001, and incorporated herein byreference. A variety of these viral serotypes and strains are availablefrom the American Type Culture Collection, Manassas, Va., or areavailable from a variety of academic or commercial sources.Alternatively, it may be desirable to synthesize sequences used inpreparing the vectors and viruses of the invention using knowntechniques, which may utilize AAV sequences which are published and/oravailable from a variety of databases. The source of the sequencesutilized in preparation of the constructs of the invention, is not alimitation of the present invention. Similarly, the selection of thespecies and serotype of AAV that provides these sequences is within theskill of the artisan and does not limit the following invention.

[0024] 2. The Minigene

[0025] For use in the present invention, the AAV sequences are typicallyin the form of a rAAV construct (e.g., a minigene or cassette) which ispackaged into a rAAV virion. At a minimum, the rAAV minigene useful inthis invention is formed by AAV inverted terminal repeat sequences(ITRs) and a heterologous molecule for delivery to a host cell. Mostsuitably, the minigene contains AAV 5′ ITRs and 3′ ITRs located 5′ and3′ to the heterologous molecule, respectively. However, in certainembodiments, it may be desirable for the minigene to contain the 5′ ITRand 3′ ITR sequences arranged in tandem, e.g., 5′ to 3′ or ahead-to-tail, or in another alternative configuration. In still otherembodiments, it may be desirable for the minigene to contain multiplecopies of the ITRs, or to have 5′ ITRs (or conversely, 3′ ITRs) locatedboth 5′ and 3′ to the heterologous molecule. The ITRs sequences may belocated immediately upstream and/or downstream of the heterologousmolecule, or there may be intervening sequences. The ITRs may beselected from AAV5, or from among the other AAV serotypes, as describedherein. Optionally, a minigene may contain 5′ ITRs from one serotype and3′ ITRs from a second serotype. The AAV sequences employed arepreferably the 145 bp cis-acting 5′ and 3′ inverted terminal repeatsequences (See, e.g., B. J. Carter, cited above). Preferably, the entiresequences encoding the ITRs are used in the molecule, although somedegree of minor modification of these sequences is permissible. Theability to modify these ITR sequences is within the skill of the art.(See, e.g., texts such as Sambrook et al, “Molecular Cloning. ALaboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York(1989); Carter et al, cited above; and K. Fisher et al., 1996 J Virol.,70:520-532). One of skill in the art can readily engineer the rAAV virusby methods known to the art (e.g., Bennett, J., et al. 1999 Proc. Natl.Acad. Sci. USA 96, 9920-9925). An example of such a molecule employed inthe present invention is a “cis-acting” plasmid containing theheterologous molecule flanked by the 5′ and 3′ AAV ITR sequences.

[0026] The heterologous molecule may be any substance which is desiredto be delivered to a cell, including, without limitation, a polypeptide,protein, enzyme, carbohydrate, chemical moiety, or nucleic acidsequences which may include oligonucleotides, RNA, and/or DNA.Preferably, for use in this invention, the heterologous molecule is aselected transgene under the control of a selected promoter and otherconventional vector regulatory components. See, e.g., U.S. Pat. Nos.5,856,152 and 5,871,982. In one embodiment, the heterologous moleculemay be a nucleic acid molecule which introduces specific geneticmodifications into human chromosomes, e.g., for correction of mutatedgenes. See, e.g., D. W. Russell & R. K. Hirata, 1998 Nat. Genet.,18:325-330.

[0027] a. The Transgene

[0028] In another desirable embodiment, the heterologous molecule is anucleic acid molecule is a transgene. As used herein, “transgene” refersto a nucleic acid sequence heterologous to the AAV sequence, encoding adesired product, e.g., a polypeptide or protein of interest, and theregulatory sequences which direct transcription and/or translationthereof in a host cell, and permit expression of the encoded product ina host cell. Suitable encoded products and regulatory sequences arediscussed in more detail below. However, the selection of theheterologous molecule delivered by the AAV minigene is not a limitationof the present invention.

[0029] In one embodiment of the method, where the ocular disorder iscaused by a mutation in a normal retinal pigment epithelium(RPE)-specific gene, the ocular cells which are the target of thetreatment method are the retinal pigment epithelial (RPE) cells. Thespecific gene which is mutated or absent in the disorder may be theRPE65 gene. Another gene which is mutated or absent in the disorder inhumans may be the arylhydrocarbon-interacting receptor protein like 1(AIPL1). Thus, the normal gene, i.e., the transgene, present in therecombinant virus is the normal, species-matched version of the mutatedgene, e.g., wildtype canine RPE65 for the treatment of canine LCA orwildtype human RPE65 for the treatment of human LCA, wildtype humanAIPL1 for the treatment of a certain type of human blinding diseases,etc. In still another embodiment, the gene can be the CRB1 (RP12) gene.In another embodiment, the transgene can be the lecithin retinalacetyltransferase (LRAT) gene. These transgenes, as well as othertransgenes useful for delivery to the eye may be obtained fromconventional sources, e.g., from university laboratories ordepositories, or synthesized from information obtained from Genbank byknown techniques.

[0030] In another embodiment of the method, where the ocular disorder iscaused by a mutation in a normal photoreceptor-specific gene, the ocularcells which are the target of the treatment method are the photoreceptorcells. The specific gene which is mutated or absent in the disorder maybe the photoreceptor-specific homeo box gene (CRX). Alternatively, thespecific gene which is mutated or absent in the disorder may be theretinal guanylate cyclase gene (GUCY2D). In still another embodiment,the transgene is a nucleotide sequence encoding RPGR Interacting Protein1 (RPGRIP1). Thus, the normal gene, i.e., the transgene, present in therecombinant adeno-associated virus is the normal, species-matchedversion of the mutated gene, e.g., wildtype murine CRX for the treatmentof the correlative murine blinding disorder or wildtype human CRX forthe treatment of the correlative human blinding disorder, wildtypechicken GUCY2D for the treatment of the correlative chicken blindingdisorder or wildtype human GUCY2D for the treatment of the correlativehuman blinding disorder, etc. These transgenes may be obtained fromconventional sources, e.g., from university laboratories ordepositories, or synthesized from information obtained from Genbank byknown techniques.

[0031] As discussed above, still other genes may be added to this list,including the LCA genes referred to as LCA3, located at chromosome 14q24and LCA5, located at chromosome 6q11-q16, among others.

[0032] Genes responsible for disorders other than LCA may also beemployed as the transgene, as suitable ocular diseases are identified.Thus, different transgene may be used in the design of similar vectorsof this invention for the treatment of disorders other than LCA. Amongthe known genes which may be absent or mutated in the blinding disordersidentified above include dystrophin, ABCR, EMP1, TIMP3, MERTCK andELOVL4. One or more of the wildtypes of these genes may be administeredto ocular cells, particularly the RPE, in the same manner as is theexemplified RPE65 for the treatment of LCA. One of skill in the art mayobtain the appropriate gene sequences and design the appropriate vectorsfor such use in view of this disclosure and in view of other informationknown to the art.

[0033] In certain situations, a different transgene may be used toencode each subunit of a protein, or to encode different peptides orproteins. This is desirable when the size of the DNA encoding theprotein subunit is large, e.g., for an immunoglobulin, theplatelet-derived growth factor, or a dystrophin protein. In order forthe cell to produce the multi-subunit protein, a cell is infected withthe recombinant virus containing each of the different subunits. Inanother embodiment, different subunits of a protein may be encoded bythe same transgene. In this case, a single transgene includes the DNAencoding each of the subunits, with the DNA for each subunit separatedby an internal ribozyme entry site (IRES). This is desirable when thesize of the DNA encoding each of the subunits is small, e.g., total ofthe DNA encoding the subunits and the IRES is less than five kilobases.Alternatively, other methods which do not require the use of an IRES maybe used for co-expression of proteins. Such other methods may involvethe use of a second internal promoter, an alternative splice signal, aco- or post-translational proteolytic cleavage strategy, among otherswhich are known to those of skill in the art.

[0034] b. Regulatory Sequences

[0035] The minigene or transgene includes appropriate sequences that areoperably linked to the nucleic acid sequences encoding the product ofinterest to promote its expression in a host cell. “Operably linked”sequences present in the minigene include both expression controlsequences (e.g. promoters) that are contiguous with the coding sequencesfor the product of interest and expression control sequences that act intrans or at a distance to control the expression of the product ofinterest. In addition to being useful in the transgene, the regulatoryelements described herein may also be used in other heterologousmolecules and the other constructs described in this application.

[0036] Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein processing and/or secretion. A great number of expressioncontrol sequences, e.g., native, constitutive, inducible and/ortissue-specific, are known in the art and may be utilized to driveexpression of the gene, depending upon the type of expression desired.

[0037] For eukaryotic cells, expression control sequences typicallyinclude a promoter, an enhancer, such as one derived from animmunoglobulin gene, SV40, cytomegalovirus, etc., and a polyadenylationsequence which may include splice donor and acceptor sites. Thepolyadenylation sequence generally is inserted following the transgenesequences and before the 3′ ITR sequence. In one embodiment, the bovinegrowth hormone polyA used.

[0038] The regulatory sequences useful in the constructs of the presentinvention may also contain an intron, desirably located between thepromoter/enhancer sequence and the gene. One possible intron sequence isalso derived from SV-40, and is referred to as the SV-40 T intronsequence. Another suitable sequence includes the woodchuck hepatitisvirus post-transcriptional element. (See, e.g., L. Wang and I. Verma,1999 Proc. Natl. Acad. Sci., USA, 96:3906-3910).

[0039] Another regulatory component of the rAAV useful in the method ofthe invention is an internal ribosome entry site (IRES). An IRESsequence, or other suitable systems as are discussed above, may be usedto produce more than one polypeptide from a single gene transcript. AnIRES (or other suitable sequence) is used to produce a protein thatcontains more than one polypeptide chain or to express two differentproteins from or within the same cell. An exemplary IRES is thepoliovirus internal ribosome entry sequence, which supports transgeneexpression in photoreceptors, RPE and ganglion cells. Preferably, theIRES is located 3′ to the transgene in the rAAV vector.

[0040] The selection of the promoter to be employed in the rAAV may bemade from among a wide number of constitutive or inducible promotersthat can express the selected transgene in an ocular. In a preferredembodiment, the promoter is cell-specific. The term “cell-specific”means that the particular promoter selected for the recombinant vectorcan direct expression of the selected transgene is a particular ocularcell type. As one example, the promoter is specific for expression ofthe transgene in RPE cells. As another example, the promoter is specificfor expression of the transgene in photoreceptor cells.

[0041] Examples of constitutive promoters which may be included in therAAV of this invention include, without limitation, the exemplified CMVimmediate early enhancer/chicken β-actin (CβA) promoter-exon 1-intron 1element of Example 1, the RSV LTR promoter/enhancer, the SV40 promoter,the CMV promoter, the dihydrofolate reductase promoter, and thephosphoglycerol kinase (PGK) promoter.

[0042] RPE-specific promoters include, for example, the RPE-65 promoter,the tissue inhibitor of metalloproteinase 3 (Timp3) promoter, and thetyrosinase promoter. Still other RPE-specific promoters are known tothose of skill in the art. See, e.g., the promoters described inInternational Patent Publication WO 00/15822.

[0043] Examples of photoreceptor specific promoters include, withoutlimitation, the rod opsin promoter, the red-green opsin promoter, theblue opsin promoter, the inter photoreceptor binding protein (IRBP)promoter and the cGMP-β-phosphodiesterase promoter. See, e.g., thepromoters described in International Patent Publication WO 98/48097.

[0044] Alternatively, an inducible promoter is employed to express thetransgene product, so as to control the amount and timing of the ocularcell's production thereof. Such promoters can be useful if the geneproduct proves to be toxic to the cell upon excessive accumulation.Inducible promoters include those known in the art and those discussedabove including, without limitation, the zinc-inducible sheepmetallothionine (MT) promoter, the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter; the T7 promoter; the ecdysoneinsect promoter; the tetracycline-repressible system; thetetracycline-inducible system; the RU486-inducible system; and therapamycin-inducible system. Any type of inducible promoter which istightly regulated and is specific for the particular target ocular celltype may be used. Other types of inducible promoters which may be usefulin this context are those which are regulated by a specificphysiological state, e.g., temperature, acute phase, a particularlydifferentiation state of the cell, or in replicating cells only.

[0045] Selection of these and other common vector and regulatoryelements are conventional and many such sequences are available. See,e.g., Sambrook et al, and references cited therein at, for example,pages 3.18-3.26 and 16.17-16.27 and Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1989). Of course, notall vectors and expression control sequences will function equally wellto express all of the transgenes of this invention. However, one ofskill in the art may make a selection among these expression controlsequences without departing from the scope of this invention. Suitablepromoter/enhancer sequences may be selected by one of skill in the artusing the guidance provided by this application. Such selection is aroutine matter and is not a limitation of the molecule or construct. Forinstance, one may select one or more expression control sequences,operably link the sequence to a transgene of interest, and insert the“minigene” comprising the expression control sequence and the transgeneinto an AAV vector. The vector may be packaged into an infectiousparticle or virion following one of the methods for packaging the rAAVtaught in the art.

[0046] C. Production of the rAAV

[0047] The rAAV virus of the invention may be constructed and producedusing the materials and methods described herein, as well as those knownto those of skill in the art. Such engineering methods used to constructany embodiment of this invention are known to those with skill innucleic acid manipulation and include genetic engineering, recombinantengineering, and synthetic techniques. See, e.g., Sambrook et al, andAusubel et al., cited above; and International Patent Application No.W095/13598. Further, methods suitable for producing a rAAV cassette inan adenoviral capsid have been described in U.S. Pat. Nos. 5,856,152 and5,871,982.

[0048] Briefly, in order to package the rAAV construct into a rAAVvirion, a host cell must contain sequences necessary to express AAV repand AAV cap or functional fragments thereof as well as helper genesessential for AAV production. For example, the rep78/52 proteins may besufficient to provide the necessary rep functions. The AAV rep and capsequences are obtained from an AAV source as identified above. The AAVrep and cap sequences may be introduced into the host cell in any mannerknown to one in the art as described above, including, withoutlimitation, transfection, electroporation, liposome delivery, membranefusion techniques, high velocity DNA-coated pellets, viral infection andprotoplast fusion. In one embodiment, the rep and cap sequences may betransfected into the host cell by one or more nucleic acid molecules andexist stably in the cell as an episome. In another embodiment, the repand cap sequences are stably integrated into the genome of the cell.Another embodiment has the rep and cap sequences transiently expressedin the host cell. For example, a useful nucleic acid molecule for suchtransfection comprises, from 5′ to 3′, a promoter, an optional spacerinterposed between the promoter and the start site of the rep genesequence, an AAV rep gene sequence, and an AAV cap gene sequence.

[0049] The rep and cap sequences, along with their expression controlsequences, may be supplied on a single vector, or each sequence may besupplied on its own vector. Preferably, the rep and cap sequences aresupplied on the same vector. Alternatively, the rep and cap sequencesmay be supplied on a vector that contains other DNA sequences that areto be introduced into the host cells. Preferably, the promoter used inthis construct may be any suitable constitutive, inducible or nativepromoters known to one of skill in the art. The molecule providing therep and cap proteins may be in any form which transfers these componentsto the host cell. Desirably, this molecule is in the form of a plasmid,which may contain other non-viral sequences, such as those for markergenes. This molecule does not contain the AAV ITRs and generally doesnot contain the AAV packaging sequences. To avoid the occurrence ofhomologous recombination, other virus sequences, particularly those ofadenovirus, are avoided in this plasmid. This plasmid is desirablyconstructed so that it may be stably transfected into a cell.

[0050] Although the molecule providing rep and cap may be transientlytransfected into the host cell, it is preferred that the host cell bestably transformed with sequences necessary to express functionalrep/cap proteins in the host cell, e.g., as an episome or by integrationinto the chromosome of the host cell. Depending upon the promotercontrolling expression of such stably transfected host cell, the rep/capproteins may be transiently expressed (e.g., through use of an induciblepromoter).

[0051] The methods employed for constructing embodiments of thisinvention are conventional genetic engineering or recombinantengineering techniques such as those described in the references above.For example, the rAAV may be produced utilizing a triple transfectionmethod using either the calcium phosphate method (Clontech) or Effectenereagent (Qiagen, Valencia, Calif.), according to manufacturer'sinstructions. See, also, Herzog et al, 1999, Nature Medic., 5(1):56-63,for the method used in the following examples, employing the plasmidwith the transgene, CPA-RPE65, a helper plasmid containing AAV rep andcap, and a plasmid supplying adenovirus helper functions of E2A, E4Orf6and VA. While this specification provides illustrative examples ofspecific constructs, using the information provided herein, one of skillin the art may select and design other suitable constructs, using achoice of spacers, promoters, and other elements, including at least onetranslational start and stop signal, and the optional addition ofpolyadenylation sites.

[0052] The rAAV virions are then produced by culturing a host cellcontaining a rAAV virus as described herein which contains a rAAVconstruct to be packaged into a rAAV virion, an AAV rep sequence and anAAV cap sequence under the control of regulatory sequences directingexpression thereof. Suitable viral helper genes, e.g., adenovirus E2A,E4Orf6 and VA, among other possible helper genes, may be provided to theculture in a variety of ways known to the art, preferably on a separateplasmid. Thereafter, the recombinant AAV virion which directs expressionof the transgene is isolated from the cell or cell culture in theabsence of contaminating helper virus or wildtype AAV.

[0053] One may easily assay whether a particular expression controlsequence is suitable for a specific transgene, and choose the expressioncontrol sequence most appropriate for expression of the desiredtransgene. For example, a target cell may be infected in vitro, and thenumber of copies of the transgene in the cell monitored by Southernblotting or quantitative polymerase chain reaction (PCR). The level ofRNA expression may be monitored by Northern blotting or quantitativereverse transcriptase (RT)-PCR; and the level of protein expression maybe monitored by Western blotting, immunohistochemistry, enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) or by the specificmethods detailed below in the examples.

[0054] In one embodiment exemplified below, a suitable recombinantvector for use in this invention is AAV-RPE65, which utilizes AAVserotype 2 ITR and capsid sequences and is described in detail inExample 1 below. This recombinant AAV contains a CMV immediate earlyenhancer/chicken β-actin (CβA) promoter-exon 1-intron 1 element followedby a poliovirus internal ribosome entry sequence (IRES), followed by thecDNA encoding the wildtype protein RPE65. However, the present inventionis not limited to this exemplary embodiment. Similar rAAV with differenttransgenes, promoters, IRES, and virus capsids may be useful in thisinvention, as described in detail above.

[0055] D. Pharmaceutical Compositions and Methods of the Invention

[0056] The recombinant AAV containing the desired transgene andcell-specific promoter for use in the target ocular cell as detailedabove is preferably assessed for contamination by conventional methodsand then formulated into a pharmaceutical composition intended forsubretinal injection. Such formulation involves the use of apharmaceutically and/or physiologically acceptable vehicle or carrier,particularly one suitable for subretinal injection, such as bufferedsaline or other buffers, e.g., HEPES, to maintain pH at appropriatephysiological levels. A variety of such known carriers are provided inInternational Patent Publication No. WO00/15822, incorporated herein byreference. If the virus is to be stored long-term, it may be frozen inthe presence of glycerol.

[0057] According to the method of this invention for treating an oculardisorder characterized by the defect or absence of a normal gene in theocular cells of a human or animal subject, the pharmaceuticalcomposition described above is administered to the subject having such ablinding disease by subretinal injection. The use of subretinalinjection as the route of delivery is a critical component of thismethod, as intravitreal administration does not enable the sametherapeutic effects. The vector and carrier cannot diffuse across themultiple cell layers in the retina to reach the RPE, when intravitrealinjection is used. Similarly, intravenous delivery is unacceptablebecause the material does not penetrate the blood-brain (blood-retinal)barrier. Because the virus does not diffuse well, topical administrationis similarly not preferred for this method. See the examples below.

[0058] An effective amount of a recombinant adeno-associated viruscarrying a nucleic acid sequence encoding the desired transgene underthe control of the cell-specific promoter sequence desirably rangesbetween about 1×10⁹ to 2×10¹² rAAV infectious units in a volume ofbetween about 150 to about 800 μl. The rAAV infectious units aremeasured as described in S. K. McLaughlin et al, 1988 J Virol., 62:1963.More desirably, an effective amount between about 1×10¹⁰ to 2×10¹¹ rAAVinfectious units in a volume of between about 250 to about 500 μl. Stillother dosages in these ranges may be selected by the attendingphysician, taking into account the physical state of the subject,preferably human, being treated, the age of the subject, the particularocular disorder and the degree to which the disorder, if progressive,has developed.

[0059] It may also be desirable to administer multiple “booster” dosagesof the pharmaceutical compositions of this invention. For example,depending upon the duration of the transgene within the ocular targetcell, one may delivered booster dosages at 6 month intervals, or yearlyfollowing the first administration. The fact that AAV-neutralizingantibodies were not generated by administration of the rAAV vector, asdiscussed in the examples below, should allow additional boosteradministrations.

[0060] Such booster dosages and the need therefor can be monitored bythe attending physicians, using, for example, the retinal and visualfunction tests and the visual behavior tests described in the examplesbelow. Other similar tests may be used to determine the status of thetreated subject over time. Selection of the appropriate tests may bemade by the attending physician. Still alternatively, the method of thisinvention may also involve injection of a larger volume ofvirus-containing solution in a single or multiple infection to allowlevels of visual function close to those found in wildtype retinas.

[0061] As is demonstrated in the examples below, an exemplary rAAVRPE65was employed in in vitro and in vivo experiments to provide evidence ofthe utility and efficacy of the methods and compositions of thisinvention. The in vitro examples demonstrated proper expression of thetransgene in an animal model of a human ocular disorder resulting inblindness. The in vivo examples demonstrated restoration of visualfunction and visual behavior by the method of this invention in a largeanimal model of a human retinopathy. The use of the exemplary vectordemonstrated in both in vitro and in vivo experiments that the defect inthe RPE65 mutant dog could be corrected by gene delivery. Vision wasrestored in this large animal model of childhood blindness. This is thefirst successful reversal of vision loss in a large animal model ofretinal degeneration. This data allow one of skill in the art to readilyanticipate that this method may be similarly used in treatment of LCA orother types of retinal disease in other subjects, including humans.

[0062] While previous studies have demonstrated that retinaldegeneration can be retarded with gene therapy techniques, the presentinvention demonstrates a definite recovery of function. In addition,while small animal studies have demonstrated histologic andelectrophysiologic correlates of visual function to be partiallypreserved, this large animal study has shown the presence of vision withregard to both physiological and behavioral measures.

EXAMPLES

[0063] As summarized, the following examples demonstrate that in threeof three eyes, in vivo transfer of wildtype RPE65 to cells of the outerretina was sufficient to restore photoreceptor function in the RPE65mutant dog. Function was not restored after intravitreal injection ofvector, a route which normally results only in transduction of ganglioncells (Dudus, L., et al. Vision Res. 39, 2545-2554 (1999)). The virustransduced RPE cells in the immediately injected quadrant, andtransduced RPE produced both wildtype RPE65 message and protein. Withoutbeing bound by theory, the inventors believe that although the rAAVvirus targets photoreceptors and other retinal neurons as well as RPEcells, and the CPA promoter is active in all these cell types, it islikely that it is the expression of the wildtype transgene in RPE cells(and not photoreceptors) that rescues the mutant phenotype. The RPEalone is responsible for, and possesses the components necessary tosupply chromophore for, rod photoreceptors, although the existence of aretinal retinoid metabolism for cones (not involving RPE65 gene product)remains plausible.

[0064] The following examples illustrate several embodiments of thisinvention. These examples are illustrative only, and do not limit thescope of the present invention.

Example 1: Virus Preparation

[0065] Recombinant AAV vector was based on pTR-UF2, a vector using the472 bp mouse rod opsin promoter to drive expression of green fluorescentprotein (GFP) (Flannery, J., et al. 1997 Proc Natl Acad Sci USA 94,6916-6921). To generate the recombinant vector, AAV-RPE65, the opsinpromoter in pTR-UF2 was replaced with a CMV immediate early enhancer(381 bp)/chicken β-actin (CβA) promoter-exon 1-intron 1 (1352 bp)element followed by a poliovirus internal ribosome entry sequence (637bp). The latter supports expression in photoreceptors, RPE and ganglioncells (Li and Hauswirth, unpublished data, 2000). The reporter/transgeneGFP was replaced with the canine RPE65 cDNA (Aguirre, G. et al, 1998Mol. Vis. 4: 23) via flanking Not I sites and the orientation andreading frame confirmed by DNA sequence analysis. Plasmid DNA containingthis construct was packaged into AAV particles employing iodixanolgradient purification followed by heparin-sepharose agarose columnchromatography as described in Hauswirth, W. W. et al, 2000 Meth.Enzymol. 316, 743-761. Vector titers were determined using an infectiouscenter assay.

[0066] Four AAV-RPE65 virus preparations were made and combined to atotal volume of 1.05 ml at 2.3×10¹¹ infectious particles/ml.Contaminating helper adenovirus and wild-type AAV, assayed by serialdilution cytopathic effect or infectious center assay, respectively wereless than six orders of magnitude lower than vector AAV.

Example 2: in Vitro Testing of an AAV Carrying the Wildtype Canine RPE65cDNA

[0067] A. RPE Cell Cultures

[0068] RPE cells from eyes of both a wildtype dog and a homozygousaffected (RPE65 mutant) dog were dissociated with 0.25% trypsin (Ray, J.et al, 1997 Curr. Eye Res. 16: 131-143) and plated at 1-2×10⁵/9 mmplastic dish. The cells were then cultured. After 48 days, confluent RPEcultures were trypsinized, subcultured and infected at 80% confluencywith 2.3×10⁷ AAV-RPE65 viral particles for 4 hours. Expression of theRPE65 transgene was assessed by immunohistochemistry 10 dayspost-infection.

[0069] B. RPE65 Immunocytochemistry and Western Analysis in Canine RPECells and Retina.

[0070] In order to evaluate the presence of the RPE65 protein, thecultured canine RPE cells were evaluated by immunocytochemistry byimmunolabelling with a rabbit anti-RPE65 peptide polyclonal antibody(generously provided by T. M. Redmond) and the nuclei were stained withpropidium iodide. For Western analysis, proteins from cultured RPE wereelectrophoresed on 12.5% SDS-polyacrylamide gel and thenelectrotransferred on nitrocellulose membrane. Immunodetection wasperformed using the anti-RPE65 antibody followed by goat anti-rabbitsecondary antibody and ¹²⁵I-protein A (Verdugo, M., et al. 1998 InvestOphthalmol Vis Sci 39, S719).

[0071] In the resulting immunohistochemical sections (not shown)wildtype retinal cells labeled uniformly and intensely with theanti-RPE65 antibody, i.e., they possessed high levels of RPE65. Incontrast, RPE65 labeling (i.e., RPE protein) was absent in untreatedRPE65 mutant cells, 60 days in culture, prior to infection withAAV-RPE65, showing only background autofluorescence. Further, lipidinclusions were apparent in the diseased RPE cells. However, within 10days of infection of the RPE65 mutant cells (60 days in culture) withAAV-RPE65, the majority of cells labeled positively with the anti-RPE65antibody, indicating presence of wildtype RPE65 protein. One cell didnot appear to have been transduced. Complementary results were observedfollowing immunohistochemistry of sections from untreated wildtypeversus mutant RPE65+ canine retinas.

[0072] Infection of the defective RPE cells by AAV-RPE65 and subsequentexpression of the wildtype RPE65 transgene were further confirmed invitro using PCR amplification and Western analysis, respectively.

[0073] PCR studies took advantage of the difference in size of thewildtype versus mutant canine RPE65 transcripts due to the 4 bp deletionin the latter. PCR amplification utilized RPE65-1 (forward) and RPE65-3(reverse) primers flanking the RPE65 mutant deletion site (Aguirre, G.et al, 1998 Mol. Vis. 4:23). PCR conditions were 94° C. (30 seconds),60° C. (30 seconds), and 72° C. (1 minute) for 34 cycles. PCR productswere separated on a 6% polyacrylamide gel. AAV-RPE65 was used aspositive control. This protocol was also used for PCR screening forshedding virus.

[0074] The PCR primers flanking this region amplified the wildtype 109bp RPE65 DNA fragment in transduced RPE cells from an affected dog.Non-transduced RPE from the same animal yielded only mutant DNA (105 bp)and normal RPE yielded only the wildtype allele (109 bp). Expression ofwildtype RPE65 in transduced RPE cells from an affected animal was alsoapparent by Western blot analysis of cell lysates. RPE65 expression wasdetected only in the transduced RPE cells; not in uninfected cells.

Example 3: in vivo Studies in the RPE65 Mutant Dog

[0075] A. Ocular Delivery

[0076] Effects of intraocular delivery of AAV-RPE65 were studied in fourRPE65 mutant dogs. For in vivo studies, virus was delivered subretinallyor intravitreally under direct surgical visualization using methodsdescribed previously (Bennett, J., et al 1999 Proc. Natl. Acad. Sci. USA96, 9920-9925 and Bennett, J. et al, 2000 Meth. Enzymol. 316, 777-789).Five eyes from three dogs (BR29, BR33 and BR47) were injected eithersubretinally or intravitreally with AAV-RPE65; the sixth eye wasuntreated (Table 1). The fourth dog (BR46) was maintained as anuntreated control.

[0077] Each 150-200 μl subretinal injection of vector (at aconcentration of 2.3×10¹¹ infectious particles/ml) created a retinaldetachment elevating approximately 35% of the total retinal area. In 2eyes (BR33 and BR47) this detachment primarily occupied thenasal-inferior quadrant; in the 3rd eye (BR29) the site wastemporal-superior. These detachments resolved spontaneously within 24hours. Animals were evaluated post-operatively for evidence of ocular orsystemic toxicity, virus exposure to extralocular tissue, virusshedding, unfavorable immune response or other untoward effects. Asdiscussed in detail below, none was found.

[0078] B. Detection of Inflammation

[0079] Eyes were evaluated clinically at regular intervals following thesurgery to identify inflammation. Humoral and intraocular antibodiesspecific to AAV capsid proteins were evaluated as described in Bennett,J., et al 1999 Proc. Natl. Acad. Sci. USA 96, 9920-9925, incorporatedherein by reference. Post-operatively, there was no evidence of ocularor systemic toxicity, or other untoward effect. Hematology and bloodchemistries revealed no evidence of systemic toxicity. Evaluation ofhumoral response prior to and post treatment revealed slightly elevatedanti-AAV capsid titers in pre-treatment serum samples, suggestingprevious exposure to AAV proteins. Antibody titers were increased in twoof the three dogs one month after exposure and in all three dogs 4months after exposure. Non-neutralizing serum antibodies directedagainst RPE65 protein also increased after intraocular exposure toAAV-RPE65.

[0080] C. Transgene Expression and Persistence

[0081] To correlate transgene expression with changed visual function,one subretinally injected eye (BR29, right eye) was surgicallyenucleated 99 days post injection. The eyecup was divided intotemporal-superior, temporal-inferior, nasal-superior, and nasal-inferiorquadrants. From each quadrant, the retina, and the pooledRPE-plus-choroid tissues, were separately harvested and dissected underRNase free conditions and rapidly frozen. Total RNA was prepared fromretina and RPE/choroid using the TRIzol Reagent kit (Life Technologies,Gaithersburg, Md.). DNA was extracted from the same tissues according tothe vendor's protocol. cDNA was amplified from total RNA using RNA PCRkit (Perkin Elmer, Foster City, Calif.) and the conditions listed above.

[0082] RPE65 expression in neural retina, RPE/choroid, and cultured RPEcells were detected. Genomic PCR demonstrates persistence of transferredviral DNA in neural retina and RPE-choroid from the injectedtemporal-superior quadrant. In other quadrants, the host DNA amplifiedpreferentially and the viral DNA amplification product is belowdetectable levels. From noninfected RPE of the affected dog, only mutantproduct amplifies, but 10 days posttransfection in vitro the normaltransgene yields the overwhelming product.

[0083] RT-PCR (figures not shown) demonstrated expression of wildtypemessage in neural retina from all 4 quadrants, but in RPE-choroid fromthe injected quadrant only. Where both products amplify, additionalbands representing heteroduplexes are also seen. The transfectedRPE/choroid from the injected quadrant expressed higher levels of thetransferred cDNA than of the mutant host gene. This was not so in otherquadrants. Although transfection of neural retina led to expression ofthe wildtype allele in all quadrants, a gradient was present in therelative intensities of the two alleles among quadrants. In the injectedquadrant, the wildtype allele yielded a much more intense band than thehost mutant allele. From the quadrant below this, the two bands wereapproximately equal in intensity. In the nasal half of the eye, themutant band predominated.

[0084] Western analysis demonstrated absence of RPE65 protein in mutantRPE cells prior to transfection, but presence of the protein afterwards.Proteins were labeled with anti-RPE65 antibody.

[0085] By PCR analyses of serum and tear fluid, there was no sign ofvirus shedding at 1 month after injection (data not shown). Reversetranscriptase (RT)-PCR on sera, conjunctiva, eyelids, the gland of thethird eyelid, and the optic nerve from the enucleated eye of BR29 werenegative for the transgene at 103 days post injection, indicating thatvirus escape to extraocular tissues was below detectable levels.

[0086] D. Retinal/Visual Function Testing

[0087] 1. Electroretinograms (ERGS)

[0088] The physiological consequences of the treatments were assessed byelectroretinography (ERG) (Banin, E., et al. 1999 Neuron 23, 549-57).Dogs were dark-adapted (overnight), premedicated with acepromazine (0.55mg/kg, IM) and atropine (0.03 mg/kg, IM) and anesthetized byintermittent ketamine (15 mg/kg, IV, repeated every 15 minutes). Pulserate, oxygen saturation and temperature were monitored throughout. Thecornea was anesthetized with topical proparacaine HCl (1%) and pupilsdilated with cyclopentolate (1%) and phenylephrine (2.5%).

[0089] Full field ERGs were recorded using a computer-based system(EPIC-XL, LKC Technologies, Inc., Gaithersburg, Md.) and Burian-Allencontact lens electrodes (Hansen Ophthalmics, Iowa city, Iowa) (Banin,E., et al. 1999 Neuron 23, 549-57). Dark-adapted luminance-responsefunctions were obtained with blue (Wratten 47A) flash stimuli spanning˜6 log units (−2.9 to +2.8 log scot-cd.s.m⁻²).

[0090] ERG b-wave amplitudes were measured conventionally from baselineor a-wave trough to positive peak; a-wave amplitude was measured frombaseline to negative peak at the maximal stimulus. For isolating conepathway function, dogs were light-adapted and 29 Hz flicker ERGs evokedwith white flash stimuli (0.4 log cd.s.m⁻²) on a background (0.8 logcd.m⁻²); amplitudes were measured between successive negative andpositive peaks and timing from stimulus to the next positive peak.Ocular axial length and pupil diameter were measured for each experimentto permit calculation of retinal illuminance.

[0091] The restoration of retinal/visual function in RPE65 mutant dogsby subretinal AAV-RPE65 was demonstrated by the results of theabove-described ERGs. A comparison of dark-adapted ERGs evoked byincreasing intensities of blue light stimuli in a control dog with ERGsto the same stimuli in RPE65 mutant dog BR33 showed the affected animalhad elevated thresholds, reduced amplitudes and waveform shape changes(i.e., b-waves but no detectable a-waves). Over a 5 log unit range ofincreasing stimulus intensity, the ERG of normal dogs responded withincreasing amplitude of bipolar cell (b-wave) and photoreceptor (a-wave)components. At all intensities these signals were dominated by rodphotoreceptor retinal pathways. Compared to normal dogs, the thresholdstimulus required to elicit an ERG response from 4 month old RPE65mutant dogs was elevated by over 4.5 log units.

[0092] Retinal function was dramatically improved in eyes treated withsubretinal AAV-RPE65, compared to pre-treatment recordings. Aftersubretinal AAV-RPE65 therapy, the mutant dog showed a vastly improvedb-wave threshold, a large increase of a- and b-wave amplitudes (althoughnot to normal levels) and an ERG waveform shape that is similar tocontrols. Responses from the right eye of BR33 had b-wave thresholdslower by ˜4 log units than pre-treatment, and appeared similar tonormal.

[0093] The details of photoreceptor function were analyzed by theamplitude and timing of the ERG photoresponses evoked by 2.8 logscot-cd.s.m⁻² flashes. Recordings from three control dogs showed ˜250 μVsaturated amplitudes peaking between 4.5 to 6 ms. Photoreceptor functionwas near noise level in three untreated eyes of RPE65 mutant dogs andtwo eyes treated with intravitreal AAV-RPE65. Photoresponses (of reducedamplitude but normal timing) were present in all three eyes thatreceived subretinal AAV-RPE65. ERG photoresponses in the threesubretinally injected eyes showed maximal amplitudes of 27, 36 and 58μV, representing ˜16% of normal (mean±SD=246±95 μV; n=7).

[0094] Small responses evoked at higher intensities lacked an a-wave.Higher energy stimuli and recording criteria that elicit, in normaldogs, saturated ERG photoresponses originating from photoreceptorsyielded no detectable signals in affected animals. A flicker ERGresponse, representing isolated cone pathway function in normals, wasabsent in affected animals. Photoresponse amplitudes in subretinallyinjected eyes were significantly different (P<0.05) than the amplitudesin untreated eyes (14±3.4 μV; n=3).

[0095] Flicker ERGs in the same eyes as described in the immediatelypreceding paragraphs demonstrated a lack of detectable cone-mediatedresponses from RPE65 mutant dogs with untreated or intravitreallytreated eyes. All eyes with subretinal AAV-RPE65 treatment recoveredcone flicker responses. Cone flicker ERGs were readily recordablepost-treatment; amplitudes ranged from 4 to 6 μV, representing ˜16% ofnormal (30±8 μV). Intravitreally injected eyes showed no difference fromuntreated eyes for all measured ERG parameters.

[0096] 2. Pupillometry

[0097] Transmission of retinal activity to higher visual pathways wasdemonstrated by pupillometry. Dogs were dark-adapted for more than 3hours and pupil responses were obtained sequentially from each eye usingfull-field green stimuli (−3.2 to +3.0 log scot-cd.m⁻²) of ˜2 secondduration. Pupils were imaged with a video camera under infraredillumination and continuously recorded on a VCR. Dynamic changes inpupil diameters were measured from single frames displayed on the videomonitor in relation to the timing of each stimulus. Pupil responses werecalculated by subtracting the smallest pupil diameter achieved within 2seconds after the stimulus onset from the diameter measured in the dark.

[0098] All tested pupils constricted in response to high intensitystimuli. Pupil response as a function of stimulus intensity showed 3.8log unit elevation of threshold (1 mm response criterion) in untreatedeyes (n=3; two eyes of BR46 and one eye of BR29) compared to normal eyes(n=3). Eyes treated with subretinal AAV (n=2; BR33 and BR47) had 0.8 logunit lower thresholds compared to untreated eyes.

[0099] A change in pupil diameter was noted in response to 2.5 logcd.m⁻² green stimulus in one eye of three representative dogs; untreated(BR46), subretinal AAV treated (BR33) and a normal control.

[0100] At a suprathreshold intensity, pupillary constriction wasgreatest in normal dogs and least in untreated RPE65 mutant dogs.Subretinally-treated eye of BR33 responded midway between normal anduntreated. The threshold intensity to reach a criterion pupillaryresponse was improved in subretinally-treated eyes compared withuntreated eyes.

[0101] Consistent with ERG and pupillometry results, at 104 dayspost-treatment, flash evoked visual cortical potentials to a series ofincreasing intensities of blue light (Wratten 47) in the dark-adaptedstate and recorded definite waveforms from the subretinally-treated eye.In contrast there were no consistent waveforms at any intensity from theeye treated intravitreally.

[0102] 3. Behavioral Testing

[0103] Qualitative visual assessment of the 3 treated animals wasperformed at 4 months post injection using an obstacle course andobservers masked to the experimental design. Visual behavior was alsodocumented by video recording. Results of behavioral testing wereconsistent with the electrophysiological results.

[0104] For example, dog BR33 was consistently (5/5 observers) scored as“normally sighted” under photopic (room lighting) conditions. Under dimred light this dog consistently avoided objects either directly in frontof her, or to her right (the side injected subretinally), butconsistently failed to avoid objects on the left (injectedintravitreally). In contrast, the untreated control affected dog, BR46walked into objects ahead of her and at either side.

[0105] Table 1 provides the data collected from the procedures performedon the eyes of four RPE65 mutant dogs. In the Table, age is recorded asdays postnatal. The abbreviation Rt is used for right eye, while left isindicated for left eye. The routes of injection are identified as SR forsubretinal injection, IV for intravitreal injection, and NI for notinjected. The doses are reported as No.×10¹⁰ infectious particles ofrecombinant AAV-RPE65 virus injected. Baseline ERGs were recorded 2weeks prior to injection. Rescue Effect was assessed by ERGs recorded 95days after injection. Positive effect is indicated by POS. NEG indicatesno effect apparent. TABLE 1 Route ERG Age at of Volume Age at RescueAnimal Day 0 Eye Injec'n Dose (μL) ERG Effect BR29 132 Rt SR 3.7 150 227POS Left NI — — 227 NEG BR33 124 Rt SR 4.6 200 219 POS Left IV 4.6 200219 NEG BR47 108 Rt SR 4.6 200 203 POS Left IV 4.6 200 203 NEG BR46 108Rt NI — — 203 NEG Left NJ — — 203 NEG

[0106] All references and documents disclosed above are incorporated byreference herein. Numerous modifications and variations of the presentinvention are included in the above-identified specification and areexpected to be obvious to one of skill in the art. Such modificationsand alterations to the compositions and processes of the presentinvention are believed to be encompassed in the scope of the claimsappended hereto.

1. A method for treating an ocular disorder characterized by the defector absence of a normal gene in the ocular cells of a subject, saidmethod comprising the step of: administering to said subject bysubretinal injection an effective amount of a recombinantadeno-associated virus carrying a nucleic acid sequence encoding saidnormal gene under the control of a promoter sequence which expresses theproduct of said gene in said ocular cells.
 2. The method according toclaim 1, wherein said ocular disorder is caused by a mutation in saidnormal retinal pigment epithelium-specific gene.
 3. The method accordingto claim 2, wherein said gene is RPE65.
 4. The method according to claim2, wherein said gene is the arylhydrocarbon-interacting receptor proteinlike 1 (AIPL1).
 5. The method according to claim 2, wherein said gene isthe CRB1 gene.
 6. The method according to claim 2, wherein said gene isthe lecithin retinal acetyltransferase gene (LRAT)
 7. The methodaccording to claim 1, wherein said ocular cells are retinal pigmentepithelial cells.
 8. The method according to claim 1, wherein saidocular disorder is caused by a mutation in said normalphotoreceptor-specific gene.
 9. The method according to claim 8, whereinsaid gene is the photoreceptor-specific homeo box gene (CRX).
 10. Themethod according to claim 8, wherein said gene is the retinal guanylatecyclase gene (GUCY2D).
 11. The method according to claim 8, wherein saidgene encodes RPGR interacting protein 1 (RPGRIP1).
 12. The methodaccording to claim 1, wherein said ocular cells are photoreceptor cells.13. The method according to claim 1, wherein said normal gene isobtained from the same subject species as the subject being treated. 14.The method according to claim 1, wherein said promoter is acell-specific promoter.
 15. The method according to claim 8, whereinsaid promoter is the chicken beta actin promoter/CMV enhancer.
 16. Themethod according to claim 1, wherein said effective amount comprises1×10⁹ to 2×10¹² rAAV infectious units in a volume of between 150 to 800μl.
 17. A composition for treatment of an ocular disorder characterizedby the defect or absence of a normal gene in the ocular cells of asubject, said composition comprising an effective amount of arecombinant adeno-associated virus carrying a nucleic acid sequenceencoding said normal gene under the control of a promoter sequence whichexpresses the product of said gene in said ocular cells, formulated witha carrier and additional components suitable for subretinal injection.18. The composition according to claim 17, wherein said normal gene is aretinal pigment epithelium-specific gene.
 19. The composition accordingto claim 18, wherein said gene is RPE65.
 20. The composition accordingto claim 18, wherein said gene is the arylhydrocarbon-interactingreceptor protein like 1 (AIPL1).
 21. The composition according to claim18, wherein said gene is the CRB1 gene.
 22. The composition according toclaim 18, wherein said gene is the lecithin retinal acetyltransferasegene (LRAT)
 23. The composition according to claim 17, wherein saidnormal gene is a photoreceptor-specific gene.
 24. The compositionaccording to claim 23, wherein said gene is the photoreceptor-specifichomeo box gene (CRX).
 25. The composition according to claim 23, whereinsaid gene is the retinal guanylate cyclase gene (GUCY2D).
 26. Thecomposition according to claim 23, wherein said gene encodes RPGRinteracting protein 1 (RPGRIP1).
 27. A method for treating an oculardisorder characterized by the defect or absence of a normal gene in theretinal pigment epithelial (RPE) cells of a subject, said methodcomprising the step of: administering to said subject by subretinalinjection an effective amount of a recombinant virus carrying a nucleicacid sequence encoding a normal retinal pigment epithelial (RPE)cell-specific gene under the control of a promoter sequence whichexpresses the product of said gene in said RPE cells.
 28. The methodaccording to claim 27, wherein said recombinant virus is anadeno-associated virus.
 29. The method according to claim 27, whereinsaid gene is the RPE65 gene.
 30. A method for treating Leber congenitalamaurosis in a subject comprising the step of administering to saidsubject by subretinal injection an effective amount of a recombinantvirus carrying a nucleic acid sequence encoding a normal gene under thecontrol of a promoter sequence which expresses the product of the genein ocular cells, wherein said cells contain a mutated version of saidgene and wherein expression of the normal gene provides to the cells theproduct necessary to restore or maintain vision in said subject.
 31. Themethod according to claim 30, wherein said ocular cells are selectedfrom the group consisting of RPE cells and photoreceptor cells.
 32. Themethod according to claim 30, wherein said promoter is cell-specific.33. The method according to claim 30, wherein said recombinant virus isa recombinant AAV carrying the normal RPE65 gene.